Novel nadh-dependent enzyme mutants to convert acetone into isopropanol

ABSTRACT

The present disclosure relates to biological processes and systems for the production of isopropanol and/or acetone utilizing modified alcohol dehydrogenases that exhibit increased activity with NADH as a cofactor. The disclosure further relates to polynucleotides and polypeptides of the modified alcohol dehydrogenases, and host cells containing the polynucleotides and expressing the polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.16/378,209, filed Apr. 8, 2019, which claims the benefit of priority toU.S. Provisional Application No. 62/653,965, filed Apr. 6, 2018, each ofwhich is hereby incorporated by reference in their entireties.

TECHNICAL FIELD

The present disclosure relates to biological processes and systems forcreating and using mutant alcohol dehydrogenase enzymes exhibitingincreased activity with NADH as a cofactor to prepare acetone orisopropanol.

STATEMENT REGARDING SEQUENCE LISTING

The sequence listing associated with this application is provided in XMLformat in lieu of a paper copy, and is hereby incorporated by referenceinto the specification. The name of the XML file containing the sequencelisting is 127125_5010_US_01_Sequence_Listing.xml. The XML file is about62,182 bytes, was created on May 30, 2023, and is being submittedelectronically via Patent Center.

BACKGROUND

Metabolic pathways for fermentative production of isopropanol may makeuse of an NADPH-dependent secondary alcohol dehydrogenase to convertacetone to isopropanol. Within the metabolic pathways there is apositive net production of reduced nicotinamide adenine dinucleotide(NADH) and consumption of reduced nicotinamide adenine dinucleotidephosphate (NADPH). Both NADH and NADPH are essential electron donors inall organisms and the cellular pool of NADPH is lower than NADH.Considerable effort has been exerted on improving the NADPH pool inorder to increase product yields dependent upon NADPH.

The present disclosure approaches the solution to increasing productyield in a different manner, by modifying the cofactor specificity ofalcohol dehydrogenase to favor utilization of NADH over NADPH.

SUMMARY OF THE DISCLOSURE

In some embodiments, the disclosure is drawn to a nucleic acid constructcomprising a polynucleotide sequence encoding a modified alcoholdehydrogenase, wherein the alcohol dehydrogenase exhibits activity withcofactor NADH as compared to an unmodified alcohol dehydrogenase; andwherein the polynucleotide sequence shares at least 85% sequenceidentity with SEQ ID NO:1.

In some embodiments, the modified alcohol dehydrogenase exhibits acofactor preference for NADH over NADPH, as compared to an unmodifiedalcohol dehydrogenase. In some embodiments, the modified alcoholdehydrogenase exhibits an increased activity for reduction of acetone toisopropanol with NADH, as compared to an unmodified alcoholdehydrogenase. In some embodiments, the modified alcohol dehydrogenaseexhibits an increased activity for oxidation of isopropanol to acetone,as compared to an unmodified alcohol dehydrogenase. In some embodiments,the modified alcohol dehydrogenase exhibits at least a 10 fold increasein activity with cofactor NADH as compared to an unmodified alcoholdehydrogenase.

In some embodiments, the modified alcohol dehydrogenase is a secondaryalcohol dehydrogenase. In some embodiments, the modified alcoholdehydrogenase is NADH-dependent. In some embodiments, the modifiedalcohol dehydrogenase is a microbial alcohol dehydrogenase. In someembodiments, the microbial alcohol dehydrogenase is a bacterial alcoholdehydrogenase. In some embodiments, the bacterial alcohol dehydrogenaseis a Clostridium beijerinckii alcohol dehydrogenase.

In some embodiments, the polynucleotide of the nucleic acid constructshares at least 90% sequence identity with SEQ ID NO:1. In someembodiments, the polynucleotide of the nucleic acid construct shares atleast 95% sequence identity with SEQ ID NO:1. In some embodiments, thepolynucleotide of the nucleic acid construct shares at least 99%sequence identity with SEQ ID NO:1

In some embodiments, the present disclosure is drawn to a polypeptidesequence comprising a modified alcohol dehydrogenase that exhibitsactivity with cofactor NADH as compared to an unmodified alcoholdehydrogenase; and wherein the modified alcohol dehydrogenase shares atleast 85% sequence identity with SEQ ID NO:2. In some embodiments, themodified alcohol dehydrogenase exhibits a cofactor preference for NADHover NADPH, as compared to an unmodified alcohol dehydrogenase. In someembodiments, the modified alcohol dehydrogenase exhibits an increasedactivity for reduction of acetone to isopropanol with NADH, as comparedto an unmodified alcohol dehydrogenase. In some embodiments, themodified alcohol dehydrogenase exhibits an increased activity foroxidation of isopropanol to acetone, as compared to an unmodifiedalcohol dehydrogenase. In some embodiments, the modified alcoholdehydrogenase exhibits at least a 10 fold increase in activity withcofactor NADH as compared to an unmodified alcohol dehydrogenase.

In some embodiments, the modified alcohol dehydrogenase is a secondaryalcohol dehydrogenase. In some embodiments, the modified alcoholdehydrogenase is NADH-dependent. In some embodiments, the modifiedalcohol dehydrogenase is a microbial alcohol dehydrogenase. In someembodiments, the microbial alcohol dehydrogenase is a bacterial alcoholdehydrogenase. In some embodiments, the bacterial alcohol dehydrogenaseis a Clostridium beijerinckii alcohol dehydrogenase.

In some embodiments, the polypeptide sequence comprises a signalpeptide. In some embodiments, the signal peptide is a secretion signal.

In some embodiments, the modified alcohol dehydrogenase shares at least90% sequence identity with SEQ ID NO:2. In some embodiments, themodified alcohol dehydrogenase shares at least 95% sequence identitywith SEQ ID NO:2. In some embodiments, the modified alcoholdehydrogenase shares at least 99% sequence identity with SEQ ID NO:2.

In some embodiments, the modified alcohol dehydrogenase sequence isselected from SEQ ID NOs: 3-38.

In some embodiments, the modified alcohol dehydrogenase sequencecomprises an amino acid substitution at one or more of the followingresidues: T38, G198, S199, Y218, I175, I173, R200, P201, C203, G244,E247, T248, K219, G243, Q251, Y267, L294, C295, K340, and K342.

In some embodiments, the modified alcohol dehydrogenase sequencecomprises an amino acid substitution at residue T38 with an H amino acidresidue. In some embodiments, the modified alcohol dehydrogenasesequence comprises an amino acid substitution at residue G198 with a D,E, G, K, N, R, S, A, or V amino acid residue. In some embodiments, themodified alcohol dehydrogenase sequence comprises an amino acidsubstitution at residue S199 with a D, G, H, I, L, N, R, S, V, C, M, orP amino acid residue. In some embodiments, wherein the modified alcoholdehydrogenase sequence comprises an amino acid substitution at residueY218 with a P, A, D, F, I, N, S, T, V, H, L, or Y amino acid residue. Insome embodiments, the modified alcohol dehydrogenase sequence comprisesan amino acid substitution at residue I173 with a V, T, A, L, F, or Yamino acid residue. In some embodiments, the modified alcoholdehydrogenase sequence comprises an amino acid substitution at residueI175 with a V or A amino acid residue. In some embodiments, the modifiedalcohol dehydrogenase sequence comprises an amino acid substitution atresidue R200 with a R, G, S, or N amino acid residue. In someembodiments, the modified alcohol dehydrogenase sequence comprises anamino acid substitution at residue P201 with a P, E, D, K, or S aminoacid residue. In some embodiments, the modified alcohol dehydrogenasesequence comprises an amino acid substitution at residue C203 with an Ror K amino acid residue. In some embodiments, the modified alcoholdehydrogenase sequence comprises an amino acid substitution at residueG244 with an A, C, M, F, I, W, L, H, or P amino acid residue. In someembodiments, the modified alcohol dehydrogenase sequence comprises anamino acid substitution at residue E247 with a D, N, Q, or H amino acidresidue. In some embodiments, the modified alcohol dehydrogenasesequence comprises an amino acid substitution at residue T248 with an S,N, A, L, M, or V amino acid residue.

In some embodiments, the modified alcohol dehydrogenase sequencecomprises one or more of the following amino acid substitutions: G198E,E247Q, E247D, E247N, Y218F, G198D, I173C, G198L, S199D, Y218P, or Y218A.

In some embodiments, the modified alcohol dehydrogenase sequencecomprises G198E and E247Q amino acid substitutions. In some embodiments,the modified alcohol dehydrogenase sequence comprises a G198E amino acidsubstitution. In some embodiments, the modified alcohol dehydrogenasesequence comprises G198E and Y218F amino acid substitutions. In someembodiments, the modified alcohol dehydrogenase sequence comprises G198Eand E247D amino acid substitutions. In some embodiments, the modifiedalcohol dehydrogenase sequence comprises G198E and E247N amino acidsubstitutions. In some embodiments, the modified alcohol dehydrogenasesequence comprises G198D and Y218F amino acid substitutions. In someembodiments, the modified alcohol dehydrogenase sequence comprises I173Cand G198E amino acid substitutions. In some embodiments, the modifiedalcohol dehydrogenase sequence comprises a G198D amino acidsubstitution. In some embodiments, the modified alcohol dehydrogenasesequence comprises G198L and S199D amino acid substitutions. In someembodiments, the modified alcohol dehydrogenase sequence comprises G198Dand Y218P amino acid substitutions. In some embodiments, the modifiedalcohol dehydrogenase sequence comprises G198E and Y218P amino acidsubstitutions. In some embodiments, the modified alcohol dehydrogenasesequence comprises G198D and Y218A amino acid substitutions.

In some embodiments, the specification is drawn to a recombinantmicroorganism comprising a nucleic acid construct of the presentdisclosure.

In some embodiments, the present disclosure is drawn to a method ofproducing a recombinant microorganism that produces a modified alcoholdehydrogenase that exhibits activity with cofactor NADH as compared toan unmodified alcohol dehydrogenase, the method comprising introducing apolynucleotide sequence encoding a polypeptide sequence of the presentdisclosure into a microorganism.

In some embodiments, the microorganism is a bacterium. In someembodiments, the bacterium is a species of Escherichia or Bacillus. Insome embodiments, the bacterium is Escherichia coli. In someembodiments, the microorganism is a fungus. In some embodiments, thefungus is a yeast. In some embodiments, the fungus is a species ofSaccharomyces, Pichia, or Aspergillus.

In some embodiments of the method, the modified alcohol dehydrogenaseexhibits a cofactor preference for NADH over NADPH, as compared to anunmodified alcohol dehydrogenase.

In some embodiments, the present disclosure is drawn to a recombinantmicroorganism expressing a polypeptide sequence comprising a modifiedalcohol dehydrogenase that exhibits activity with cofactor NADH ascompared to an unmodified alcohol dehydrogenase; and wherein themodified alcohol dehydrogenase shares at least 85% sequence identitywith SEQ ID NO:2. In some embodiments of the recombinant microorganism,the modified alcohol dehydrogenase exhibits a cofactor preference forNADH over NADPH, as compared to an unmodified alcohol dehydrogenase. Insome embodiments of the recombinant microorganism, the modified alcoholdehydrogenase exhibits an increased activity for reduction of acetone toisopropanol, as compared to an unmodified alcohol dehydrogenase. In someembodiments of the recombinant microorganism, the modified alcoholdehydrogenase exhibits an increased activity for oxidation ofisopropanol to acetone, as compared to an unmodified alcoholdehydrogenase. In some embodiments of the recombinant microorganism, themodified alcohol dehydrogenase exhibits at least a 10 fold increase inactivity with cofactor NADH as compared to an unmodified alcoholdehydrogenase.

In some embodiments of the recombinant microorganism, the modifiedalcohol dehydrogenase is a secondary alcohol dehydrogenase. In someembodiments of the recombinant microorganism, the modified alcoholdehydrogenase is NADH-dependent. In some embodiments of the recombinantmicroorganism, the modified alcohol dehydrogenase is a microbial alcoholdehydrogenase. In some embodiments of the recombinant microorganism, themicrobial alcohol dehydrogenase is a bacterial alcohol dehydrogenase. Insome embodiments of the recombinant microorganism, the bacterial alcoholdehydrogenase is a Clostridium bejerinckii alcohol dehydrogenase.

In some embodiments of the recombinant microorganism, the polypeptidesequence comprises a signal peptide. In some embodiments of therecombinant microorganism, the signal peptide is a secretion signal.

In some embodiments of the recombinant microorganism, the modifiedalcohol dehydrogenase shares at least 90% sequence identity with SEQ IDNO:2. In some embodiments of the recombinant microorganism, the modifiedalcohol dehydrogenase shares at least 95% sequence identity with SEQ IDNO:2. In some embodiments of the recombinant microorganism, the modifiedalcohol dehydrogenase shares at least 99% sequence identity with SEQ IDNO:2.

In some embodiments of the recombinant microorganism, the modifiedalcohol dehydrogenase sequence is selected from SEQ ID NOs: 3-38.

In some embodiments of the recombinant microorganism, the modifiedalcohol dehydrogenase sequence comprises an amino acid substitution atone or more of the following residues: T38, G198, S199, Y218, I175,I173, R200, P201, C203, G244, E247, T248, K219, G243, Q251, Y267, L294,C295, K340, and K342.

In some embodiments of the recombinant microorganism, the modifiedalcohol dehydrogenase sequence comprises an amino acid substitution atresidue T38 with an H amino acid residue. In some embodiments of therecombinant microorganism, the modified alcohol dehydrogenase sequencecomprises an amino acid substitution at residue G198 with a D, E, G, K,N, R, S, A, or V amino acid residue. In some embodiments of therecombinant microorganism, the modified alcohol dehydrogenase sequencecomprises an amino acid substitution at residue S199 with a D, G, H, I,L, N, R, S, V, C, M, or P amino acid residue. In some embodiments of therecombinant microorganism, wherein the modified alcohol dehydrogenasesequence comprises an amino acid substitution at residue Y218 with a P,A, D, F, I, N, S, T, V, H, L, or Y amino acid residue. In someembodiments of the recombinant microorganism, the modified alcoholdehydrogenase sequence comprises an amino acid substitution at residueI173 with a V, T, A, L, F, or Y amino acid residue. In some embodimentsof the recombinant microorganism, the modified alcohol dehydrogenasesequence comprises an amino acid substitution at residue I175 with a Vor A amino acid residue. In some embodiments of the recombinantmicroorganism, the modified alcohol dehydrogenase sequence comprises anamino acid substitution at residue R200 with a R, G, S, or N amino acidresidue. In some embodiments of the recombinant microorganism, themodified alcohol dehydrogenase sequence comprises an amino acidsubstitution at residue P201 with a P, E, D, K, or S amino acid residue.In some embodiments of the recombinant microorganism, the modifiedalcohol dehydrogenase sequence comprises an amino acid substitution atresidue C203 with an R or K amino acid residue. In some embodiments ofthe recombinant microorganism, the modified alcohol dehydrogenasesequence comprises an amino acid substitution at residue G244 with an A,C, M, F, I, W, L, H, or P amino acid residue. In some embodiments of therecombinant microorganism, the modified alcohol dehydrogenase sequencecomprises an amino acid substitution at residue E247 with a D, N, Q, orH amino acid residue. In some embodiments of the recombinantmicroorganism, the modified alcohol dehydrogenase sequence comprises anamino acid substitution at residue T248 with an S, N, A, L, M, or Vamino acid residue.

In some embodiments of the recombinant microorganism, the modifiedalcohol dehydrogenase sequence comprises one or more of the followingamino acid substitutions: G198E, E247Q, E247D, E247N, Y218F, G198D,I173C, G198L, S199D, Y218P, or Y218A.

In some embodiments of the recombinant microorganism, the modifiedalcohol dehydrogenase sequence comprises G198E and E247Q amino acidsubstitutions. In some embodiments of the recombinant microorganism, themodified alcohol dehydrogenase sequence comprises a G198E amino acidsubstitution. In some embodiments of the recombinant microorganism, themodified alcohol dehydrogenase sequence comprises G198E and Y218F aminoacid substitutions. In some embodiments of the recombinantmicroorganism, the modified alcohol dehydrogenase sequence comprisesG198E and E247D amino acid substitutions. In some embodiments of therecombinant microorganism, the modified alcohol dehydrogenase sequencecomprises G198E and E247N amino acid substitutions. In some embodimentsof the recombinant microorganism, the modified alcohol dehydrogenasesequence comprises G198D and Y218F amino acid substitutions. In someembodiments of the recombinant microorganism, the modified alcoholdehydrogenase sequence comprises I173C and G198E amino acidsubstitutions. In some embodiments of the recombinant microorganism, themodified alcohol dehydrogenase sequence comprises a G198D amino acidsubstitution. In some embodiments of the recombinant microorganism, themodified alcohol dehydrogenase sequence comprises G198L and S199D aminoacid substitutions. In some embodiments of the recombinantmicroorganism, the modified alcohol dehydrogenase sequence comprisesG198D and Y218P amino acid substitutions. In some embodiments of therecombinant microorganism, the modified alcohol dehydrogenase sequencecomprises G198E and Y218P amino acid substitutions. In some embodimentsof the recombinant microorganism, the modified alcohol dehydrogenasesequence comprises G198D and Y218A amino acid substitutions.

In some embodiments of the recombinant microorganism, the modifiedalcohol dehydrogenase is NADH-dependent

In some embodiments, the modified alcohol dehydrogenase may comprise anyone or more mutations described in FIG. 1 . In some embodiments, therecombinant microorganism may express any one or more polypeptidescomprising any one or more of the mutations described in FIG. 1 . Insome embodiments. The nucleic acid construction may encode a polypeptidesequence comprising any one or more of the mutations described in FIG. 1.

In some embodiments, the modified alcohol dehydrogenase may comprise anyone or more mutations described in FIG. 2 . In some embodiments, therecombinant microorganism may express any one or more polypeptidescomprising any one or more of the mutations described in FIG. 2 . Insome embodiments. The nucleic acid construction may encode a polypeptidesequence comprising any one or more of the mutations described in FIG. 2.

In some embodiments, the modified alcohol dehydrogenase may comprise anyone or more mutations described in FIG. 3 . In some embodiments, therecombinant microorganism may express any one or more polypeptidescomprising any one or more of the mutations described in FIG. 3 . Insome embodiments. The nucleic acid construction may encode a polypeptidesequence comprising any one or more of the mutations described in FIG. 3.

In some embodiments, the modified alcohol dehydrogenase may comprise anyone or more mutations described in FIG. 4 . In some embodiments, therecombinant microorganism may express any one or more polypeptidescomprising any one or more of the mutations described in FIG. 4 . Insome embodiments. The nucleic acid construction may encode a polypeptidesequence comprising any one or more of the mutations described in FIG. 4.

In some embodiments, the modified alcohol dehydrogenase may comprise anyone or more mutations described in FIG. 5 . In some embodiments, therecombinant microorganism may express any one or more polypeptidescomprising any one or more of the mutations described in FIG. 5 . Insome embodiments. The nucleic acid construction may encode a polypeptidesequence comprising any one or more of the mutations described in FIG. 5.

In some embodiments, the modified alcohol dehydrogenase may comprise anyone or more mutations described in FIG. 6 . In some embodiments, therecombinant microorganism may express any one or more polypeptidescomprising any one or more of the mutations described in FIG. 6 . Insome embodiments. The nucleic acid construction may encode a polypeptidesequence comprising any one or more of the mutations described in FIG. 6.

In some embodiments, the modified alcohol dehydrogenase may comprise anyone or more mutations described in FIG. 7 . In some embodiments, therecombinant microorganism may express any one or more polypeptidescomprising any one or more of the mutations described in FIG. 7 . Insome embodiments. The nucleic acid construction may encode a polypeptidesequence comprising any one or more of the mutations described in FIG. 7.

In some embodiments, the modified alcohol dehydrogenase may comprise anyone or more mutations described in FIG. 8 . In some embodiments, therecombinant microorganism may express any one or more polypeptidescomprising any one or more of the mutations described in FIG. 8 . Insome embodiments. The nucleic acid construction may encode a polypeptidesequence comprising any one or more of the mutations described in FIG. 8.

BRIEF DESCRIPTION OF THE DRAWINGS

Aspects of the disclosure are illustrated in the drawings, in which:

FIG. 1 illustrates a tabulation of enzyme kinetics for the use of NADHor Acetone in an assay for alcohol dehydrogenase (ADH) activity in amodified (G198D, S199V, P201E, and Y218A) ADH from each of Clostridiumbeijerinckii and Clostridium autoethanogenum.

FIG. 2 illustrates a tabulation of enzyme kinetics for the use of NADPHor Acetone in an assay for alcohol dehydrogenase (ADH) activity in amodified (G198D, S199V, P201E, and Y218A) ADH from each of Clostridiumbeijerinckii and Clostridium autoethanogenum.

FIG. 3 illustrates a tabulation of enzyme kinetics for the use of NADHor Acetone in an assay for alcohol dehydrogenase (ADH) activity inmultiple modified ADHs from Clostridium beijerinckii.

FIG. 4 illustrates a tabulation of enzyme kinetics for the use of NADPHor Acetone in an assay for alcohol dehydrogenase (ADH) activity inmultiple modified ADHs from Clostridium beijerinckii.

FIG. 5 illustrates a tabulation of Clostridium beijerinckii alcoholdehydrogenase (ADH) mutants with an affirmative or negative indicationas to whether the mutants accept NADH or NADPH cofactors.

FIG. 6 illustrates a tabulation of Clostridium beijerinckii alcoholdehydrogenase (ADH) mutants that were tested for NADH or NADPH cofactoracceptance.

FIG. 7 illustrates a tabulation of the amino acid residues ofClostridium beijerinckii alcohol dehydrogenase (ADH) which may bemutated to cause a switch in preferential usage of NADH to NADPH as acofactor, and in some aspects the residues which may be utilized.

FIG. 8 depicts a list of positive tested mutants for NADH acceptance.

FIG. 9 illustrates a depiction of the tabulation of Clostridiumbeijerinckii alcohol dehydrogenase (ADH) alignment with possiblemutations based on alignments of ADH with the NADPH motif.

FIG. 10 illustrates a depiction of the tabulation of Clostridiumbeijerinckii alcohol dehydrogenase (ADH) alignment with possiblemutations based on alignments of ADH with the NADH motif.

DETAILED DESCRIPTION Definitions

The following definitions and abbreviations are to be used for theinterpretation of the disclosure.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a three-carbon compound”includes a plurality of such three-carbon compounds and reference to“the microorganism” includes reference to one or more microorganisms,and so forth.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having, “contains,” “containing,” or any othervariation thereof, are intended to cover a non-exclusive inclusion. Acomposition, mixture, process, method, article, or apparatus thatcomprises a list of elements is not necessarily limited to only thoseelements but may include other elements not expressly listed or inherentto such composition, mixture, process, method, article, or apparatus.Further, unless expressly stated to the contrary, “or” refers to aninclusive “or” and not to an exclusive “or.”

The terms “about” and “around,” as used herein to modify a numericalvalue, indicate a close range surrounding that explicit value. If “X”were the value, “about X” or “around X” would indicate a value from 0.9Xto 1.1X, or, in some embodiments, a value from 0.95X to 1.05X. Anyreference to “about X” or “around X” specifically indicates at least thevalues X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X,and 1.05X. Thus, “about X” and “around X” are intended to teach andprovide written description support for a claim limitation of, e.g.,“0.98X.”

As used herein, the terms “microbial,” “microbial organism,” and“microorganism” include any organism that exists as a microscopic cellthat is included within the domains of archaea, bacteria or eukaryota,the latter including yeast and filamentous fungi, protozoa, algae, orhigher Protista. Therefore, the term is intended to encompassprokaryotic or eukaryotic cells or organisms having a microscopic sizeand includes bacteria, archaea, and eubacteria of all species as well aseukaryotic microorganisms such as yeast and fungi. Also included arecell cultures of any species that can be cultured for the production ofa chemical.

As described herein, in some embodiments, the recombinant microorganismsare prokaryotic microorganisms. In some embodiments, the prokaryoticmicroorganisms are bacteria. “Bacteria”, or “eubacteria”, refers to adomain of prokaryotic organisms. Bacteria include at least elevendistinct groups as follows: (1) Gram-positive (gram+) bacteria, of whichthere are two major subdivisions: (1) high G+C group (Actinomycetes,Mycobacteria, Micrococcus, others) (2) low G+C group (Bacillus,Clostridia, Lactobacillus, Staphylococci, Streptococci, Mycoplasmas);(2) Proteobacteria, e.g., Purple photosynthetic+non-photosyntheticGram-negative bacteria (includes most “common” Gram-negative bacteria);(3) Cyanobacteria, e.g., oxygenic phototrophs; (4) Spirochetes andrelated species; (5) Planctomyces; (6) Bacteroides, Flavobacteria; (7)Chlamydia; (8) Green sulfur bacteria; (9) Green non-sulfur bacteria(also anaerobic phototrophs); (10) Radioresistant micrococci andrelatives; (11) Thermotoga and Thermosipho thermophiles.

“Gram-negative bacteria” include cocci, nonenteric rods, and entericrods. The genera of Gram-negative bacteria include, for example,Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella,Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella,Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter,Agrobacterium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium,Chlamydia, Rickettsia, Treponema, and Fusobacterium.

“Gram positive bacteria” include cocci, nonsporulating rods, andsporulating rods. The genera of gram positive bacteria include, forexample, Actinomyces, Bacillus, Clostridium, Corynebacterium,Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus,Nocardia, Staphylococcus, Streptococcus, and Streptomyces.

The term “recombinant microorganism” and “recombinant host cell” areused interchangeably herein and refer to microorganisms that have beengenetically modified to express or to overexpress endogenous enzymes, toexpress heterologous enzymes, such as those included in a vector, in anintegration construct, or which have an alteration in expression of anendogenous gene. By “alteration” it is meant that the expression of thegene, or level of a RNA molecule or equivalent RNA molecules encodingone or more polypeptides or polypeptide subunits, or activity of one ormore polypeptides or polypeptide subunits is up regulated or downregulated, such that expression, level, or activity is greater than orless than that observed in the absence of the alteration. For example,the term “alter” can mean “inhibit,” but the use of the word “alter” isnot limited to this definition. It is understood that the terms“recombinant microorganism” and “recombinant host cell” refer not onlyto the particular recombinant microorganism but to the progeny orpotential progeny of such a microorganism. Because certain modificationsmay occur in succeeding generations due to either mutation orenvironmental influences, such progeny may not, in fact, be identical tothe parent cell, but are still included within the scope of the term asused herein.

The term “expression” with respect to a gene sequence refers totranscription of the gene and, as appropriate, translation of theresulting mRNA transcript to a protein. Thus, as will be clear from thecontext, expression of a protein results from transcription andtranslation of the open reading frame sequence. The level of expressionof a desired product in a host cell may be determined on the basis ofeither the amount of corresponding mRNA that is present in the cell, orthe amount of the desired product encoded by the selected sequence. Forexample, mRNA transcribed from a selected sequence can be quantitated byqRT-PCR or by Northern hybridization (see Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press(1989)). Protein encoded by a selected sequence can be quantitated byvarious methods, e.g., by ELISA, by assaying for the biological activityof the protein, or by employing assays that are independent of suchactivity, such as western blotting or radioimmunoassay, using antibodiesthat recognize and bind the protein. See Sambrook et al., 1989, supra.

The term “polynucleotide” is used herein interchangeably with the term“nucleic acid” and refers to an organic polymer composed of two or moremonomers including nucleotides, nucleosides or analogs thereof,including but not limited to single stranded or double stranded, senseor antisense deoxyribonucleic acid (DNA) of any length and, whereappropriate, single stranded or double stranded, sense or antisenseribonucleic acid (RNA) of any length, including siRNA. The term“nucleotide” refers to any of several compounds that consist of a riboseor deoxyribose sugar joined to a purine or a pyrimidine base and to aphosphate group, and that are the basic structural units of nucleicacids. The term “nucleoside” refers to a compound (as guanosine oradenosine) that consists of a purine or pyrimidine base combined withdeoxyribose or ribose and is found especially in nucleic acids. The term“nucleotide analog” or “nucleoside analog” refers, respectively, to anucleotide or nucleoside in which one or more individual atoms have beenreplaced with a different atom or with a different functional group.Accordingly, the term polynucleotide includes nucleic acids of anylength, DNA, RNA, analogs and fragments thereof. A polynucleotide ofthree or more nucleotides is also called nucleotide oligomer oroligonucleotide.

It is understood that the polynucleotides described herein include“genes” and that the nucleic acid molecules described herein include“vectors” or “plasmids.” Accordingly, the term “gene”, also called a“structural gene” refers to a polynucleotide that codes for a particularsequence of amino acids, which comprise all or part of one or moreproteins or enzymes, and may include regulatory (non-transcribed) DNAsequences, such as promoter sequences, which determine for example theconditions under which the gene is expressed. The transcribed region ofthe gene may include untranslated regions, including introns,5′-untranslated region (UTR), and 3′-UTR, as well as the codingsequence.

The terms nucleic acid “constructs” or “vectors” and like terms shouldbe taken broadly to include any nucleic acid (including DNA, cDNA andRNA) suitable for use as a vehicle to transfer genetic material into acell. The terms should be taken to include plasmids, viruses (includingbacteriophage), cosmids and artificial chromosomes. Constructs orvectors may include one or more regulatory elements, an origin ofreplication, a multicloning site and/or a selectable marker. In oneparticular embodiment, the constructs or vectors are adapted to allowexpression of one or more genes encoded by the construct or vectorNucleic acid constructs or vectors include naked nucleic acids as wellas nucleic acids formulated with one or more agents to facilitatedelivery to a cell (for example, liposome-conjugated nucleic acid, anorganism in which the nucleic acid is contained). The vectors may beused for cloning or expression of nucleic acids and for transformationof microorganisms to produce recombinant microorganisms. The vectors mayinclude one or more nucleic acids encoding one or more alcoholdehydrogenase enzyme of the disclosure.

The term “enzyme” as used herein refers to any substance that catalyzesor promotes one or more chemical or biochemical reactions, which usuallyincludes enzymes totally or partially composed of a polypeptide orpolypeptides, but can include enzymes composed of a different moleculeincluding polynucleotides

As used herein, the term “chimeric” or “recombinant” when describing anucleic acid sequence or a protein sequence refers to a nucleic acid, ora protein sequence, that links at least two heterologouspolynucleotides, or two heterologous polypeptides, into a singlemacromolecule, or that re-arranges one or more elements of at least onenatural nucleic acid or protein sequence. For example, the term“recombinant” can refer to an artificial combination of two otherwiseseparated segments of sequence, e.g., by chemical synthesis or by themanipulation of isolated segments of nucleic acids by geneticengineering techniques.

As used herein, a “synthetic nucleotide sequence” or “syntheticpolynucleotide sequence” is a nucleotide sequence that is not known tooccur in nature or that is not naturally occurring. Generally, such asynthetic nucleotide sequence will comprise at least one nucleotidedifference when compared to any other naturally occurring nucleotidesequence.

As used herein, the term “nucleic acid” refers to a polymeric form ofnucleotides of any length, either ribonucleotides ordeoxyribonucleotides, or analogs thereof. This term refers to theprimary structure of the molecule, and thus includes double- andsingle-stranded DNA, as well as double- and single-stranded RNA. It alsoincludes modified nucleic acids such as methylated and/or capped nucleicacids, nucleic acids containing modified bases, backbone modifications,and the like. The terms “nucleic acid” and “nucleotide sequence” areused interchangeably.

As used herein, the term “gene” refers to any segment of DNA associatedwith a biological function. Thus, genes include, but are not limited to,coding sequences and/or the regulatory sequences required for theirexpression. Genes can also include non-expressed DNA segments that, forexample, form recognition sequences for other proteins. Genes can beobtained from a variety of sources, including cloning from a source ofinterest or synthesizing from known or predicted sequence information,and may include sequences designed to have desired parameters.

As used herein, the term “homologous” or “homologue” or “ortholog” isknown in the art and refers to related sequences that share a commonancestor or family member and are determined based on the degree ofsequence identity. The terms “homology,” “homologous,” “substantiallysimilar” and “corresponding substantially” are used interchangeablyherein. They refer to nucleic acid fragments wherein changes in one ormore nucleotide bases do not affect the ability of the nucleic acidfragment to mediate gene expression or produce a certain phenotype.These terms also refer to modifications of the nucleic acid fragments ofthe instant disclosure such as deletion or insertion of one or morenucleotides that do not substantially alter the functional properties ofthe resulting nucleic acid fragment relative to the initial, unmodifiedfragment. It is therefore understood, as those skilled in the art willappreciate, that the disclosure encompasses more than the specificexemplary sequences. These terms describe the relationship between agene found in one species, subspecies, variety, cultivar or strain andthe corresponding or equivalent gene in another species, subspecies,variety, cultivar or strain. For purposes of this disclosure homologoussequences are compared. “Homologous sequences” or “homologues” or“orthologs” are thought, believed, or known to be functionally related.A functional relationship may be indicated in any one of a number ofways, including, but not limited to: (a) degree of sequence identityand/or (b) the same or similar biological function. Preferably, both (a)and (b) are indicated. Homology can be determined using softwareprograms readily available in the art, such as those discussed inCurrent Protocols in Molecular Biology (F. M. Ausubel et al., eds.,1987) Supplement 30, section 7.718, Table 7.71. Some alignment programsare MacVector (Oxford Molecular Ltd, Oxford, U.K.), ALIGN Plus(Scientific and Educational Software, Pennsylvania) and AlignX (VectorNTI, Invitrogen, Carlsbad, Calif.). Another alignment program isSequencher (Gene Codes, Ann Arbor, Mich.), using default parameters.

As used herein, the term “nucleotide change” refers to, e.g., nucleotidesubstitution, deletion, and/or insertion, as is well understood in theart. For example, mutations contain alterations that produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded protein or how the proteins are made.

As used herein, the term “protein modification” refers to, e.g., aminoacid substitution, amino acid modification, deletion, and/or insertion,as is well understood in the art.

As used herein, the term “at least a portion” or “fragment” of a nucleicacid or polypeptide means a portion having the minimal sizecharacteristics of such sequences, or any larger fragment of the fulllength molecule, up to and including the full length molecule. Afragment of a polynucleotide of the disclosure may encode a biologicallyactive portion of a genetic regulatory element. A biologically activeportion of a genetic regulatory element can be prepared by isolating aportion of one of the polynucleotides of the disclosure that comprisesthe genetic regulatory element and assessing activity as describedherein. Similarly, a portion of a polypeptide may be 4 amino acids, 5amino acids, 6 amino acids, 7 amino acids, and so on, going up to thefull length polypeptide. The length of the portion to be used willdepend on the particular application. A portion of a nucleic acid usefulas a hybridization probe may be as short as 12 nucleotides; in someembodiments, it is 20 nucleotides. A portion of a polypeptide useful asan epitope may be as short as 4 amino acids. A portion of a polypeptidethat performs the function of the full-length polypeptide wouldgenerally be longer than 4 amino acids.

Variant polynucleotides also encompass sequences derived from amutagenic and recombinogenic procedure such as DNA shuffling. Strategiesfor such DNA shuffling are known in the art. See, for example, Stemmer(1994) PNAS 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameriet al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol.Biol. 272:336-347; Zhang et al. (1997) PNAS 94:4504-4509; Crameri et al.(1998) Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.For PCR amplifications of the polynucleotides disclosed herein,oligonucleotide primers can be designed for use in PCR reactions toamplify corresponding DNA sequences from cDNA or genomic DNA extractedfrom any organism of interest. Methods for designing PCR primers and PCRcloning are generally known in the art and are disclosed in Sambrook etal. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold SpringHarbor Laboratory Press, Plainview, N.Y.). See also Innis et al., eds.(1990) PCR Protocols: A Guide to Methods and Applications (AcademicPress, New York); Innis and Gelfand, eds. (1995) PCR Strategies(Academic Press, New York); and Innis and Gelfand, eds. (1999) PCRMethods Manual (Academic Press, New York). Known methods of PCR include,but are not limited to, methods using paired primers, nested primers,single specific primers, degenerate primers, gene-specific primers,vector-specific primers, partially-mismatched primers, and the like.

The term “primer” as used herein refers to an oligonucleotide which iscapable of annealing to the amplification target allowing a DNApolymerase to attach, thereby serving as a point of initiation of DNAsynthesis when placed under conditions in which synthesis of primerextension product is induced, i.e., in the presence of nucleotides andan agent for polymerization such as DNA polymerase and at a suitabletemperature and pH. The (amplification) primer is preferably singlestranded for maximum efficiency in amplification. Preferably, the primeris an oligodeoxyribonucleotide. The primer must be sufficiently long toprime the synthesis of extension products in the presence of the agentfor polymerization. The exact lengths of the primers will depend on manyfactors, including temperature and composition (A/T vs. G/C content) ofprimer. A pair of bi-directional primers consists of one forward and onereverse primer as commonly used in the art of DNA amplification such asin PCR amplification.

As used herein, “promoter” refers to a DNA sequence capable ofcontrolling the expression of a coding sequence or functional RNA. Thepromoter sequence consists of proximal and more distal upstreamelements, the latter elements often referred to as enhancers.Accordingly, an “enhancer” is a DNA sequence that can stimulate promoteractivity, and may be an innate element of the promoter or a heterologouselement inserted to enhance the level or specificity of a promoter.Promoters may be derived in their entirety from a native gene, or becomposed of different elements derived from different promoters found innature, or even comprise synthetic DNA segments. It is understood bythose skilled in the art that different promoters may direct theexpression of a gene in different cell types, or at different stages ofdevelopment, or in response to different environmental conditions. It isfurther recognized that since in most cases the exact boundaries ofregulatory sequences have not been completely defined, DNA fragments ofsome variation may have identical promoter activity.

As used herein, a “constitutive promoter” is a promoter which is activeunder most conditions and/or during most development stages. There areseveral advantages to using constitutive promoters in expression vectorsused in biotechnology, such as: high level of production of proteinsused to select transgenic cells or organisms; high level of expressionof reporter proteins or scoreable markers, allowing easy detection andquantification; high level of production of a transcription factor thatis part of a regulatory transcription system; production of compoundsthat requires ubiquitous activity in the organism; and production ofcompounds that are required during all stages of development.Non-limiting exemplary constitutive promoters include, CaMV 35Spromoter, opine promoters, ubiquitin promoter, alcohol dehydrogenasepromoter, etc.

As used herein, a “non-constitutive promoter” is a promoter which isactive under certain conditions, in certain types of cells, and/orduring certain development stages. For example, cell type specific, celltype preferred, inducible promoters, and promoters under developmentcontrol are non-constitutive promoters. Examples of promoters underdevelopmental control include promoters that preferentially initiatetranscription in certain tissues.

As used herein, “inducible” or “repressible” promoter is a promoterwhich is under chemical or environmental factors control. Examples ofenvironmental conditions that may affect transcription by induciblepromoters include anaerobic conditions, certain chemicals, the presenceof light, acidic or basic conditions, etc.

As used herein, the phrases “recombinant construct”, “expressionconstruct”, “chimeric construct”, “construct”, and “recombinant DNAconstruct” are used interchangeably herein. A recombinant constructcomprises an artificial combination of nucleic acid fragments, e.g.,regulatory and coding sequences that are not found together in nature.For example, a chimeric construct may comprise regulatory sequences andcoding sequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. Such constructmay be used by itself or may be used in conjunction with a vector. If avector is used then the choice of vector is dependent upon the methodthat will be used to transform host cells as is well known to thoseskilled in the art. For example, a plasmid vector can be used. Theskilled artisan is well aware of the genetic elements that must bepresent on the vector in order to successfully transform, select andpropagate host cells comprising any of the isolated nucleic acidfragments of the disclosure. The skilled artisan will also recognizethat different independent transformation events will result indifferent levels and patterns of expression (Jones et al., (1985) EMBOJ. 4:2411-2418; De Almeida et al., (1989) Mol. Gen. Genetics 218:78-86),and thus that multiple events must be screened in order to obtain linesdisplaying the desired expression level and pattern. Such screening maybe accomplished by Southern analysis of DNA, Northern analysis of mRNAexpression, immunoblotting analysis of protein expression, or phenotypicanalysis, among others. Vectors can be plasmids, viruses,bacteriophages, pro-viruses, phagemids, transposons, artificialchromosomes, and the like, that replicate autonomously or can integrateinto a chromosome of a host cell. A vector can also be a naked RNApolynucleotide, a naked DNA polynucleotide, a polynucleotide composed ofboth DNA and RNA within the same strand, a poly-lysine-conjugated DNA orRNA, a peptide-conjugated DNA or RNA, a liposome-conjugated DNA, or thelike, that is not autonomously replicating. As used herein, the term“expression” refers to the production of a functional end-product e.g.,an mRNA or a protein (precursor or mature).

As used herein, the term “non-naturally occurring,” when used inreference to a microorganism, organism, or enzyme activity of thedisclosure, is intended to mean that the microorganism organism orenzyme has at least one genetic alteration not normally found in anaturally occurring strain of the referenced species, includingwild-type strains of the referenced species. Genetic alterationsinclude, for example, modifications introducing expressible nucleicacids encoding metabolic polypeptides, other nucleic acid additions,nucleic acid deletions and/or other functional disruption of themicroorganism's genetic material. Such modifications include, forexample, coding regions and functional fragments thereof, forheterologous, homologous, or both heterologous and homologouspolypeptides for the referenced species. Additional modificationsinclude, for example, non-coding regulatory regions in which themodifications alter expression of a gene or operon. Exemplarynon-naturally occurring microorganism or enzyme activity includes thehydroxylation activity described above. As used herein, the term“operably linked” refers to the association of nucleic acid sequences ona single nucleic acid fragment so that the function of one is regulatedby the other.

The term “exogenous” as used herein with reference to various molecules,e.g., polynucleotides, polypeptides, enzymes, etc., refers to moleculesthat are not normally or naturally found in and/or produced by a givenyeast, bacterium, organism, microorganism, or cell in nature.

On the other hand, the term “endogenous” or “native” as used herein withreference to various molecules, e.g., polynucleotides, polypeptides,enzymes, etc., refers to molecules that are normally or naturally foundin and/or produced by a given yeast, bacterium, organism, microorganism,or cell in nature.

The term “heterologous” as used herein in the context of a modified hostcell refers to various molecules, e.g., polynucleotides, polypeptides,enzymes, etc., wherein at least one of the following is true: (a) themolecule(s) is/are foreign (“exogenous”) to (i.e., not naturally foundin) the host cell; (b) the molecule(s) is/are naturally found in (e.g.,is “endogenous to”) a given host microorganism or host cell but iseither produced in an unnatural location or in an unnatural amount inthe cell; and/or (c) the molecule(s) differ(s) in nucleotide or aminoacid sequence from the endogenous nucleotide or amino acid sequence(s)such that the molecule differing in nucleotide or amino acid sequencefrom the endogenous nucleotide or amino acid as found endogenously isproduced in an unnatural (e.g., greater than naturally found) amount inthe cell.

The term “homolog,” as used herein with respect to an original enzyme orgene of a first family or species, refers to distinct enzymes or genesof a second family or species which are determined by functional,structural, or genomic analyses to be an enzyme or gene of the secondfamily or species which corresponds to the original enzyme or gene ofthe first family or species. Homologs most often have functional,structural, or genomic similarities. Techniques are known by whichhomologs of an enzyme or gene can readily be cloned using genetic probesand PCR. Identity of cloned sequences as homologs can be confirmed usingfunctional assays and/or by genomic mapping of the genes.

A protein has “homology” or is “homologous” to a second protein if theamino acid sequence encoded by a gene has a similar amino acid sequenceto that of the second gene. Alternatively, a protein has homology to asecond protein if the two proteins have “similar” amino acid sequences.Thus, the term “homologous proteins” is intended to mean that the twoproteins have similar amino acid sequences. In certain instances, thehomology between two proteins is indicative of its shared ancestry,related by evolution. The terms “homologous sequences” or “homologs” arethought, believed, or known to be functionally related. A functionalrelationship may be indicated in any one of a number of ways, including,but not limited to: (a) degree of sequence identity and/or (b) the sameor similar biological function. Preferably, both (a) and (b) areindicated. The degree of sequence identity may vary, but in oneembodiment, is at least 50% (when using standard sequence alignmentprograms known in the art), at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least about 91%,at least about 92%, at least about 93%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least 98.5%, or at least about 99%, or at least 99.5%, or at least99.8%, or at least 99.9%. Homology can be determined using softwareprograms readily available in the art, such as those discussed inCurrent Protocols in Molecular Biology (F. M. Ausubel et al., eds.,1987) Supplement 30, section 7.718, Table 7.71. Some alignment programsare MacVector (Oxford Molecular Ltd, Oxford, U.K.) and ALIGN Plus(Scientific and Educational Software, Pennsylvania). Other non-limitingalignment programs include Sequencher (Gene Codes, Ann Arbor, Mich.),AlignX, and Vector NTI (Invitrogen, Carlsbad, Calif.). A similarbiological function may include, but is not limited to: catalyzing thesame or similar enzymatic reaction; having the same or similarselectivity for a substrate or co-factor; having the same or similarstability; having the same or similar tolerance to various fermentationconditions (temperature, pH, etc.); and/or having the same or similartolerance to various metabolic substrates, products, by-products,intermediates, etc. The degree of similarity in biological function mayvary, but in one embodiment, is at least 1%, at least 2%, at least 3%,at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, atleast 9%, at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least about 91%, at leastabout 92%, at least about 93%, at least about 94%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, or at least98.5%, or at least about 99%, or at least 99.5%, or at least 99.8%, orat least 99.9%, according to one or more assays known to one skilled inthe art to determine a given biological function.

The term “variant” refers to any polypeptide or enzyme described herein.A variant also encompasses one or more components of a multimer,multimers comprising an individual component, multimers comprisingmultiples of an individual component (e.g., multimers of a referencemolecule), a chemical breakdown product, and a biological breakdownproduct. These variants may be referred to herein as “functionallyequivalent variants” A functionally equivalent variant of a protein or apeptide includes those proteins or peptides that share at least 40%,preferably 50%, preferably 60%, preferably 70%, preferably 75%,preferably 80%, preferably 85%, preferably 90%, preferably 95% orgreater amino acid identity with the protein or peptide identified andhas substantially the same function as the peptide or protein ofinterest. Such variants include within their scope fragments of aprotein or peptide wherein the fragment comprises a truncated form ofthe polypeptide wherein deletions may be from 1 to 5, to 10, to 15, to20, to 25 amino acids, and may extend from residue 1 through 25 ateither terminus of the polypeptide, and wherein deletions may be of anylength within the region; or may be at an internal location.Functionally equivalent variants of the specific polypeptides hereinshould also be taken to include polypeptides expressed by homologousgenes in other species of bacteria, for example as exemplified in theprevious paragraph. In particular, non-limiting embodiments, an enzymemay be a “variant” relative to a reference enzyme by virtue ofalteration(s) in any part of the polypeptide sequence encoding thereference enzyme. A variant of a reference enzyme can have enzymeactivity of at least 10%, at least 30%, at least 50%, at least 80%, atleast 90%, at least 100%, at least 105%, at least 110%, at least 120%,at least 130% or more in a standard assay used to measure enzymeactivity of a preparation of the enzyme.

The alcohol dehydrogenase enzymes of the disclosure are referred toherein to have “increased specificity” for one substrate over another.This is intended to mean that the alcohol dehydrogenase has increasedspecificity for one substrate relative to another, compared to the wildtype alcohol dehydrogenase. It should not be taken to necessarily inferthat an alcohol dehydrogenase of the disclosure has a higher specificityfor a particular substrate compared to the wild type alcoholdehydrogenase, although this may be the case in some embodiments.Additionally, the term should not be taken to mean that an alcoholdehydrogenase of the disclosure has absolute specificity for aparticular substrate over another, although this may be the case in someembodiments, and includes at least a preference for a particularsubstrate over another substrate.

“Increased specificity”, “higher specificity”, “preferentialspecificity” or like terms, when used in relation to an NADH or NADPHco-factor, refers to the degree of affinity with which a co-factor bindsto an alcohol dehydrogenase during a reaction. It should not be taken tomean that an alcohol dehydrogenase and a co-factor have absolutespecificity, although this may be the case, and includes at least apreference for the binding between a particular alcohol dehydrogenaseand one co-factor over another co-factor.

As used herein, the term “cofactor specificity” or “cofactor preference”is a measure of the specificity of an enzyme for one cofactor overanother. Thus the methods of the present invention may be used to alterthe cofactor preference of the target enzyme, such that the preferencefor the less favored cofactor is increased by 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 90%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%,22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%,36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99, 100%, 125%, 150%, 175%, 200%,250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, 1,000%,2,000%, 3,000%, 4,000%, 5,000%. For target proteins that prefer NADPH asa cofactor, it would be desirable to alter the cofactor specificity ofthe target enzyme (e.g., an alcohol dehydrogenase) to a cofactor that ismore readily available, such as NADH.

Alcohol Dehydrogenase (ADH)

The disclosure identifies modifications of alcohol dehydrogenase thatresult in an increased or preferential specificity for one or moresubstrates and/or cofactors over other substrates and/or cofactors. Thisdisclosure contemplates that the modification and use of a wide varietyof alcohol dehydrogenases from a wide variety of organisms, includingalcohol dehydrogenases that exhibit activity towards primary and/orsecondary alcohols, and uses NADH and/or NADPH as substrate/cofactor (EC1.1.1.1 or EC 1.1.1.2). In one embodiment, the wt alcohol dehydrogenaseis selected from any one of the microorganisms set forth in the presentdisclosure.

The ADH from wt strains C. beijerinckii are known to exhibit enzymaticactivity on acetone and butyraldehyde in the presence of cofactor NADPH;however, the strains exhibited no measurable enzymatic activity onacetone and butyraldehyde in the presence of cofactor NADH. See Ismaielet al. (1993. J. Bac. 175(16):5097-5105).

In some embodiments, alcohol dehydrogenases of the present disclosureshare at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 99%, or at least 100% sequence identity with SEQ ID NO:2.

In some embodiments, alcohol dehydrogenases of the present disclosureshare at least about 50%, at least about 55%, at least about 60%, atleast about 65%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least 9 about 5%, atleast about 99%, or at least about 100% sequence identity with SEQ IDNO:2.

In some embodiments, alcohol dehydrogenases of the present disclosureare encoded by polynucleotide sequences that share at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 99%, or atleast 100% sequence identity with SEQ ID NO:1.

In some embodiments, alcohol dehydrogenases of the present disclosureare encoded by polynucleotide sequences that share at least about 50%,at least about 55%, at least about 60%, at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least 9 about 5%, at least about 99%, or at leastabout 100% sequence identity with SEQ ID NO:1.

In some embodiments, alcohol dehydrogenases of the present disclosurecomprise at least one mutation/modification compared to thecorresponding wt alcohol dehydrogenase.

In some embodiments, the modified alcohol dehydrogenase comprises anamino acid substitution at one or more of the following residues: T38,G198, S199, Y218, I175, I173, R200, P201, C203, G244, E247, T248, K219,G243, Q251, Y267, L294, C295, K340, and K342.

In some embodiments, the modified alcohol dehydrogenase comprises anamino acid substitution at T38 with an H amino acid residue.

In some embodiments, the modified alcohol dehydrogenase comprises anamino acid substitution at residue G198 with a D, E, G, K, N, R, S, A,or V amino acid residue.

In some embodiments, the modified alcohol dehydrogenase comprises anamino acid substitution at residue S199 with a D, G, H, I, L, N, R, S,V, C, M, or P amino acid residue.

In some embodiments, the modified alcohol dehydrogenase comprises anamino acid substitution at residue Y218 with a P, A, D, F, I, N, S, T,V, Y, H, L, or Y amino acid residue.

In some embodiments, the modified alcohol dehydrogenase comprises anamino acid substitution at residue I173 with a V, T, A, L, T, F, or Yamino acid residue.

In some embodiments, the modified alcohol dehydrogenase comprises anamino acid substitution at residue I175 with a V or A amino acidresidue.

In some embodiments, the modified alcohol dehydrogenase comprises anamino acid substitution at residue R200 with a R, G, S, or N amino acidresidue.

In some embodiments, the modified alcohol dehydrogenase comprises anamino acid substitution at residue P201 with a P, E, D, K, or S aminoacid residue.

In some embodiments, the modified alcohol dehydrogenase comprises anamino acid substitution at residue C203 with an R or K amino acidresidue.

In some embodiments, the modified alcohol dehydrogenase comprises anamino acid substitution at residue G244 with an A, C, M, F, I, W, L, H,or P amino acid residue.

In some embodiments, the modified alcohol dehydrogenase comprises anamino acid substitution at residue E247 with a D, N, Q, or H amino acidresidue.

In some embodiments, the modified alcohol dehydrogenase comprises anamino acid substitution at residue T248 with an S, N, A, L, M, or Vamino acid residue.

In some embodiments, the modified alcohol dehydrogenase comprises one ormore of the following amino acid substitutions: I173C, G198E, G218F,Y218P, Y218A, Y218W, S199D, S199L, G244A, G244L, G244C, E247D, E247N,E247Q, G198D, S199G, S199V, P201E, P201R, I175R, and Y218D.

In some embodiments, the modified alcohol dehydrogenase comprises aminoacid substitution G198E and only one additional substitution selectedfrom the following amino acid substitutions: I173C, G218F, Y218P, Y218A,Y218W, S199D, S199L, G244A, G244L, G244C, E247D, E247N, E247Q, G198D,S199G, S199V, P201E, P201R, I175R, and Y218D.

In some embodiments, the modified alcohol dehydrogenase comprises aminoacid substitution G198E and at least one additional substitutionselected from the following amino acid substitutions: I173C, G218F,Y218P, Y218A, Y218W, S199D, S199L, G244A, G244L, G244C, E247D, E247N,E247Q, G198D, S199G, S199V, P201E, P201R, I175R, and Y218D.

In some embodiments, the modified alcohol dehydrogenase comprises fewerthan 4 amino acid substitutions. In some embodiments, the modifiedalcohol dehydrogenase comprises fewer than 3 amino acid substitutions.In some embodiments, the modified alcohol dehydrogenase comprises 2amino acid substitutions. In some embodiments, the modified alcoholdehydrogenase comprises 3 amino acid substitutions. In some embodiments,the modified alcohol dehydrogenase comprises 4 amino acid substitutions.In some embodiments, the modified alcohol dehydrogenase comprises atleast 1 amino acid substitution. In some embodiments, the modifiedalcohol dehydrogenase comprises at least 2 amino acid substitutions. Insome embodiments, the modified alcohol dehydrogenase comprises at least3 amino acid substitutions. In some embodiments, the modified alcoholdehydrogenase comprises at least 4 amino acid substitutions.

In some embodiments, the modified alcohol dehydrogenase comprises aG198E amino acid substitution. In some embodiments, the modified alcoholdehydrogenase comprises a G198D amino acid substitution. In someembodiments, the modified alcohol dehydrogenase comprises an amino acidsubstitution at G198E and I173C. In some embodiments, the modifiedalcohol dehydrogenase comprises an amino acid substitution at G198E andY218F. In some embodiments, the modified alcohol dehydrogenase comprisesan amino acid substitution at G198E and Y218P. In some embodiments, themodified alcohol dehydrogenase comprises an amino acid substitution atG198E and Y218A. In some embodiments, the modified alcohol dehydrogenasecomprises an amino acid substitution at G198E and Y218W. In someembodiments, the modified alcohol dehydrogenase comprises an amino acidsubstitution at G198E and S199D. In some embodiments, the modifiedalcohol dehydrogenase comprises an amino acid substitution at G198E andS199I. In some embodiments, the modified alcohol dehydrogenase comprisesan amino acid substitution at G198E and G244A. In some embodiments, themodified alcohol dehydrogenase comprises an amino acid substitution atG198E and G244L. In some embodiments, the modified alcohol dehydrogenasecomprises an amino acid substitution at G198E and G244C. In someembodiments, the modified alcohol dehydrogenase comprises an amino acidsubstitution at G198E and E247D. In some embodiments, the modifiedalcohol dehydrogenase comprises an amino acid substitution at G198E andE247N. In some embodiments, the modified alcohol dehydrogenase comprisesan amino acid substitution at G198E and E247Q. In some embodiments, themodified alcohol dehydrogenase comprises an amino acid substitution atG198D and S199I. In some embodiments, the modified alcohol dehydrogenasecomprises an amino acid substitution at G198D and S199G. In someembodiments, the modified alcohol dehydrogenase comprises an amino acidsubstitution at G198D and Y218F. In some embodiments, the modifiedalcohol dehydrogenase comprises an amino acid substitution at G198D andY218P. In some embodiments, the modified alcohol dehydrogenase comprisesan amino acid substitution at G198D and Y218W. In some embodiments, themodified alcohol dehydrogenase comprises an amino acid substitution atG198D and Y218A. In some embodiments, the modified alcohol dehydrogenasecomprises an amino acid substitution at G198D, S199V, P201E, and Y218A.In some embodiments, the modified alcohol dehydrogenase comprises anamino acid substitution at G198D S199V, and P201R. In some embodiments,the modified alcohol dehydrogenase comprises an amino acid substitutionat G198L and S199D. In some embodiments, the modified alcoholdehydrogenase comprises an amino acid substitution at I175R, G198L,S199D, and Y218D.

In some embodiments, the amino acid substitutions of the presentdisclosure are relative to SEQ ID NO:2. In some embodiments, the aminoacid substitutions of the present disclosure are relative to wt alcoholdehydrogenases. In some embodiments, the amino acid substitutions of thepresent disclosure are relative to wt microbial alcohol dehydrogenases.In some embodiments, the amino acid substitutions of the presentdisclosure are relative to wt bacterial alcohol dehydrogenases.

In some embodiments, the modified alcohol dehydrogenase exhibits anincreased specificity for NADH over NADPH as a cofactor, exhibits theability to utilize NADH as a cofactor, or exhibits NADH dependence as acofactor.

In some embodiments, the modified alcohol dehydrogenase exhibits anincreased specificity for NADH over NADPH, as compared to acorresponding unmodified alcohol dehydrogenase. In some embodiments, themodified alcohol dehydrogenase exhibits an increased specificity forNADH over NADPH by at least 5%, at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at last 100%, at least200%, at least 300%, at least 400%, at least 500%, at least 600%, atleast 700%, at least 800%, at least 900%, or at least 1,000% as comparedto a corresponding unmodified alcohol dehydrogenase.

In some embodiments, the modified alcohol dehydrogenase exhibits anincreased specificity for NADH over NADPH by at least about 5%, at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 95%, at last about 100%, at least about 200%,at least about 300%, at least about 400%, at least about 500%, at leastabout 600%, at least about 700%, at least about 800%, at least about900%, or at least about 1,000% as compared to a corresponding unmodifiedalcohol dehydrogenase.

In some embodiments, the modified alcohol dehydrogenase exhibits anincreased specificity for NADH over NADPH at a ratio of 1:10, 2:10,3:10, 4:10, 5:10, 6:10, 7:10, 8:10, 9:10, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1,7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1,100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, or1,000:1.

In some embodiments, the modified alcohol dehydrogenase exhibits anincreased specificity for NADH over NADPH at a ratio of at least 1:10,at least 2:10, at least 3:10, at least 4:10, at least 5:10, at least6:10, at least 7:10, at least 8:10, at least 9:10, at least 1:1, atleast 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1, atleast 7:1, at least 8:1, at least 9:1, at least 10:1, at least 15:1, atleast 20:1, at least 25:1, at least 30:1, at least 35:1, at least 40:1,at least 45:1, at least 50:1, at least 100:1, at least 200:1, at least300:1, at least 400:1, at least 500:1, at least 600:1, at least 700:1,at least 800:1, at least 900:1, or at least 1,000:1.

In some embodiments, the modified alcohol dehydrogenase exhibits anincreased specificity for NADH over NADPH at a ratio of at least about1:10, at least about 2:10, at least about 3:10, at least about 4:10, atleast about 5:10, at least about 6:10, at least about 7:10, at leastabout 8:10, at least about 9:10, at least about 1:1, at least about 2:1,at least about 3:1, at least about 4:1, at least about 5:1, at leastabout 6:1, at least about 7:1, at least about 8:1, at least about 9:1,at least about 10:1, at least about 15:1, at least about 20:1, at leastabout 25:1, at least about 30:1, at least about 35:1, at least about40:1, at least about 45:1, at least about 50:1, at least about 100:1, atleast about 200:1, at least about 300:1, at least about 400:1, at leastabout 500:1, at least about 600:1, at least about 700:1, at least about800:1, at least about 900:1, or at least about 1,000:1.

In some embodiments, the modified alcohol dehydrogenase utilizes bothNADH and NADPH as a cofactor/substrate. In some embodiments, themodified alcohol dehydrogenase is a microbial alcohol dehydrogenase. Insome embodiments, the modified alcohol dehydrogenase is a bacterialalcohol dehydrogenase. In some embodiments, the modified alcoholdehydrogenase is an Archaea alcohol dehydrogenase. In some embodiments,the modified alcohol dehydrogenase is a fungal alcohol dehydrogenase.

Nucleic Acids

The present disclosure relates to novel alcohol dehydrogenases, itfurther relates to nucleic acids encoding the alcohol dehydrogenases andnucleic acid constructs and vectors comprising the nucleic acids.

The present disclosure contemplates codon optimization of nucleic acidsencoding an alcohol dehydrogenase of the disclosure, for any type oforganisms.

In some embodiments, the disclosure contemplates nucleic acid vectorsand/or constructs comprising one or more nucleic acids encoding one ormore alcohol dehydrogenase of the disclosure. In some embodiments,nucleic acids of the disclosure may remain extrachromosomal upontransformation of a microorganism or may be adapted for integration intothe genome of the microorganism. Accordingly, nucleic acids of thedisclosure may include additional nucleotide sequences adapted to assistintegration (for example, a region which allows for homologousrecombination and targeted integration into the host genome) or stableexpression and replication of an extrachromosomal construct (forexample, origin of replication, promoter and other regulatorysequences).

In some embodiments, nucleic acids encoding one or more alcoholdehydrogenase of the disclosure will comprise a promoter adapted topromote expression of the one or more enzymes encoded by the nucleicacids. In one embodiment, the promoter is a constitutive promoter thatis preferably highly active under appropriate fermentation conditions.Inducible promoters could also be used. In preferred embodiments, thepromoter is selected from the group comprising Wood-Ljungdahl genecluster or an arabinose inducible pBAD promoter. It will be appreciatedby those of skill in the art that other promoters which can directexpression, preferably a high level of expression under appropriatefermentation conditions, would be effective as alternatives to theexemplified embodiments.

In some embodiments, nucleic acids and nucleic acid constructs,including expression constructs/vectors of the disclosure may beconstructed using any number of techniques standard in the art. Forexample, chemical synthesis, site directed mutagenesis, or recombinanttechniques may be used. Such techniques are described, for example, inSambrook et al (Molecular Cloning: A laboratory manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) Suitablevectors for use in the disclosure will be appreciated by those ofordinary skill in the art.

Microorganisms

The microorganisms of the disclosure may be prepared from a parentalmicroorganism using any number of techniques known in the art,including, for example, site directed mutagenesis techniques tointroduce the desired mutation(s) into an alcohol dehydrogenase genenative to a parental microorganism, or other recombinant technologies tointroduce one or more nucleic acid encoding one or more alcoholdehydrogenase of the disclosure into a parental microorganism.

In one embodiment, one or more exogenous nucleic acid encoding one ormore alcohol dehydrogenase is introduced into a parental microorganismand replaces one or more alcohol dehydrogenase gene native to theparental microorganism. In another embodiment, one or more exogenousnucleic acid encoding one or more alcohol dehydrogenase of thedisclosure is introduced to a parental microorganism and issupplementary to an alcohol dehydrogenase gene native to the parentalmicroorganism. In other embodiments, one or more exogenous nucleic acidis introduced into a parental microorganism to introduce one or moredesired mutation into one or more alcohol dehydrogenase gene native tothe parental microorganism. In another embodiment, one or more exogenousnucleic acid encoding one or more alcohol dehydrogenase is introducedinto a parental microorganism, and one or more mutation is introduced toone or more alcohol dehydrogenase gene native to the parentalmicroorganism to reduce or knock out its expression and activity.

In one embodiment, a microorganism of the disclosure is prepared from aparental microorganism using recombinant technology. For example, aparental microorganism is transformed with one or more exogenous nucleicacid encoding an alcohol dehydrogenase of the disclosure, or one or morenucleic acid adapted to introduce a desired mutation to a native alcoholdehydrogenase gene in the parental microorganism. An exogenous nucleicacid may remain extrachromosomal upon transformation of the parentmicroorganism or may integrate into the genome of the parentmicroorganism (in one embodiment to replace a native alcoholdehydrogenase gene, or introduce a mutation into a native alcoholdehydrogenase gene). Accordingly, they may include additional nucleotidesequences adapted to assist integration (for example, a region whichallows for homologous recombination and targeted integration into thehost genome) or expression and replication of an extrachromosomalconstruct (for example, origin of replication, promoter and otherregulatory elements or sequences), as described herein.

In one embodiment, transformation (including transduction ortransfection) of a microorganism may be achieved by electroporation,ultrasonication, polyethylene glycol-mediated transformation, chemicalor natural competence, or conjugation. Suitable transformationtechniques are described for example in, Sambrook J, Fritsch E F,Maniatis T: Molecular Cloning: A laboratory Manual, Cold Spring HarbourLaboratory Press, Cold Spring Harbour, 1989.

In one embodiment, one or more exogenous nucleic acids may be deliveredto a parental microorganism as naked nucleic acids or may be formulatedwith one or more agents to facilitate the transformation process (forexample, liposome-conjugated nucleic acid, an organism in which thenucleic acid is contained). The one or more nucleic acids may be DNA,RNA, or combinations thereof, as is appropriate. Restriction inhibitorsmay be used in certain embodiments; see, for example Murray, N. E. etal. (2000) Microbial. Molec. Biol. Rev. 64, 412.)

In one embodiment, the recombinant microorganism is chosen from a groupof microorganisms comprising bacteria, Archaea, and fungi.

In one embodiment, the recombinant microorganism is chosen from thegenera Clostridium, Acetobacterium, Moorella, Butyribacterium, Blautia,Oxobacter, Thermoanaerobacter, Escherichia, Klebsiella, Zymomonas,Citrobacter, Enterobacter, Salmonella, Serratia, Lactobacillus.Lactococcus. Enterococcus, Pediococcus, Streptococcus, Saccharomyces,Pichia, Candida hansemila, Yarrowia, Rhodotorula, Rhizopus,Trichosporon, Lipomyces, Aspergillus, trichoderma, Exophila, Mucor,Cladosporium, Phanerochaete, Cladophialophora, Paecilomyces,Scedosporium, Ophistoma, Bacillus, Oligotropha, Pseudomonas,Carbophilus, Hydrogenophaga, Mycobacterium, Zavarzinia, Cupravidus,Senechocystis, Chloroflexus, Methylomonas, Methylobacter, Methylococcus,Methylomicrobium, Methylosphera, Methylocaldum, Methylocystis,Methylosinus, Methanobacterium, Methanococcus, Methanogenium,Methanosarcina, Methanosphaera, Methanothermobacter, Methanotrix,Corynebacterium, Acinetobacter, Actinomyces, Bacteroides, Burkholderia,Brevibacterium, Pyrococcus, Geobacter, Geobacillus, Paenibacillus,Mycobacterium, Rhodopseudomonas, Thermatoga, Thermoanaerobacter,Streptomyces, Rhodobacter, Rhodococcus, Peptococcus, Bifidobacterium,Propionibacterium, Fusobacterium, Campylobacter, Veillonella, Aquincola,Arthrobacter, Moraxella, and Psychrobacter.

In one embodiment the organism is chosen from the group ofcarboxydotrophic acetogenic microorganisms, the group of Enterobacteria,the group of Lactobacillus, the group of fungi and yeasts, the group ofaerobic carboxydotrophes, the group of aerobic CO₂ fixing organisms, thegroup of methylotrophes, and the group of methanogens.

In one embodiment, the microorganism is a carboxydotrophic acetogenselected from the group comprising Clostridium autoethanogenum,Clostridium ljungdahlii, Clostridium ragsdalei, Clostridiumcarboxidivorans, Clostridium drakei, Clostridium scatologenes,Clostridium coskatii, Clostridium aceticum, Clostridium magnum,Clostridium sp., Butyribacterium limosum, Butyribacteriummethylotrophicum, Acetobacterium woodii, Alkalibaculum bacchii, Blautiaproducta, Eubacterium limosum, Moorella thermoacetica, Moorellathermautotrophica, Oxobacier pfennigii, and Thermoanaerobacter kiuvi. Inone embodiment the microorganism is Clostridium autoethanogenum, orClostridium ljungdahlii.

In one embodiment, the microorganism is a microorganism selected fromthe group comprising Clostridium acetobutylicum, Clostridiumbeijerinckii, Clostridium saccharobutylicum, Clostridiumsaccharaoperbutylacetonicum. In one embodiment the microorganism isClostridium acetobutylicum, or Clostridium beijerinckii.

In one embodiment, the microorganism is an Enterobacteria selected fromthe group comprising Escherichia, Klebsiella, Zymomonas, Citrobacter,Enterobacter, Salmonella, and Serratia. In one embodiment themicroorganism is Escherichia coli, Zymomonas mobilis, Klebsiellapneumonia, Klebsiella oxtoca, Enterobacter cloacae, or Serratamarcescens.

In one embodiment, the microorganism is a Lactobacillus selected fromthe group comprising Lactobacillus, Lactococcus, Enterococcus,Pediococcus, and Streptococcus. In one embodiment the microorganism isLactobacillus brevis, Enterococcus faecalis, or Lactococcus lactis.

In one embodiment, the microorganism is a fungi or yeast selected fromthe group comprising Saccharomyces. Pichia, Candida hansemila, Yarrowia,Rhodotorula, Rhizopus, Trichosporon, Lipomyces and from the groupcomprising Aspergillus, Trichoderma, Exophila, Mucor, Cladosporium,Phanerochaete, Cladophialophora, Paecilomyces, Scedosporium, Ophistoma.In one embodiment the microorganism is Saccharomyces cerevisae. Candidatropicalis, Candida albicans or Yarrowia lipolytica. In one embodimentthe microorganism is Aspergillus niger, or Trichoderma reesei.

In one embodiment, the microorganism is an aerobic carboxydotrophselected from the group comprising Bacillus, Oligotropha, Pseudomonas,Carbophilus, Hydrogenophaga, Mycobacterium, and Zavarzinia. In oneembodiment the microorganism is Oligotropha carboxidovorans, Carbophiluscarboxidus, Hydrogenophaga pseudoflava, Mycobacterium sp., Pseudomonascarboxydohydrogena, Pseudomonas sp., Zavarzinia compransoris or Bacillusschlegelli. In one embodiment, the microorganism is an aerobic CO₂fixing organism selected from the group comprising Cupravidus,Senechocystis, and Chloroflexus. In one embodiment the microorganism isCupravidus necator, Synechocystis sp., or Chloroflexus aurantiacus.

In one embodiment, the microorganism is a methylotroph selected from thegroup comprising Methylomonas, Methylobacter, Methylococcus,Methylomicrobium, Methylosphera, Methylocaldum, Methylocystis, orMethylosinus. In one embodiment the microorganism is Methylococcuscapsulatus, or Methylosinus trichosporium.

In one embodiment, the microorganism is a methanogen selected from thegroup comprising Methanobacterium, Methanococcus, Methanogenium,Methanosarcina, Methanosphaera, Methanothermobacter, and Methanotrix. Inone embodiment the microorganism is Methanothermobacter marburgensis, orMethanosarcina barkeri.

The present disclosure provides a method for producing isopropanoland/or acetone by microbial fermentation of a substrate using arecombinant microorganisms of the disclosure. In some embodiments, theisopropanol and/or acetone are collected or isolated. In someembodiments, the isopropanol and/or acetone are purified.

Example 1: Modification of Two Alcohol Dehydrogenases from Clostridium

Previous work indicated a cofactor preference change for NADH over NADPHin Clostridium autoethanogenum with G198D, S199V, P201E, and Y218A (DVEAmutant) mutations in the C. autoethanogenum alcohol dehydrogenase. (SeePCT Publication No. WO2013152236A1, and Protein Engineering, Design &Selection. 2015. 28.8:251-258). The alcohol dehydrogenase fromClostridium beijerinckii shares 86% identity and 94% similarity to C.autoethanogenum. The C. autoethanogenum DVEA mutant of alcoholdehydrogenase was found to convert acetone to isopropanol utilizing NADHand/or NADPH.

The DVEA mutant was recreated in the alcohol dehydrogenase from C.beijerinckii, and compared against the DVEA mutant from C.autoethanogenum for NADH utilization (FIG. 1 ) and NADPH utilization(FIG. 2 ). The DVEA mutant from C. beijerinckii exhibited asignificantly decreased catalytic efficiency for at least the NADH, ascompared with the significantly higher catalytic efficiency exhibited bythe DVEA mutant from C. autoethanogenum.

Example 2: Modifications of Alcohol Dehydrogenases from Clostridiumbeijerinckii

The C. beijerinckii alcohol dehydrogenase was utilized to make numerousmutations that resulted in between 1 and 4 amino acid substitutions ineach of the alcohol dehydrogenase mutants (FIG. 6 ). FIG. 5 indicatesthe cofactor acceptance status for each of the tested mutants,indicating whether NADH and/or NADPH activity was present. FIG. 8indicates the mutants that were capable of utilizing NADH, and FIG. 3and FIG. 4 reveal the enzyme kinetics data for NADH and NADPH acceptancefor a subset of the mutants from FIG. 8 . FIG. 7 indicates the residuesin the alcohol dehydrogenase that are hot spots for substitutions thatcan confer NADH acceptance, with some of the possible mutationsindicated for some of the residues. FIG. 9 and FIG. 10 indicate thealignment with possible mutations based upon alignments with the alcoholdehydrogenase and the NADH and NADPH motifs.

The previous work with alcohol dehydrogenase from C. autoethanogenumrequired several mutations (DVEA mutant; See PCT Publication No.WO2013152236A1). However, FIG. 3 demonstrates NADH cofactor acceptancein mutants that only required two amino acid substitutions in thealcohol dehydrogenase. Comparing the results of FIG. 3 (C. beijerinckii)with the C. autoethanogenum of FIG. 1 makes it abundantly clear thatfewer substitutions in the alcohol dehydrogenases can be made to yieldenzyme activity that surpasses that of the C. autoethanogenum DVEAmutant.

EMBODIMENTS OF THE DISCLOSURE

1. A nucleic acid construct comprising a polynucleotide sequenceencoding a modified alcohol dehydrogenase, wherein the alcoholdehydrogenase exhibits activity with cofactor NADH as compared to anunmodified alcohol dehydrogenase; and wherein the polynucleotidesequence shares at least 85% sequence identity with SEQ ID NO:1.

2. A nucleic acid construct of embodiment 1, wherein the modifiedalcohol dehydrogenase exhibits a cofactor preference for NADH overNADPH, as compared to an unmodified alcohol dehydrogenase.

3. The nucleic acid construct of embodiment 1, wherein the modifiedalcohol dehydrogenase exhibits an increased activity for reduction ofacetone to isopropanol with NADH, as compared to an unmodified alcoholdehydrogenase.

4. The nucleic acid construct of embodiment 1, wherein the modifiedalcohol dehydrogenase exhibits an increased activity for oxidation ofisopropanol to acetone, as compared to an unmodified alcoholdehydrogenase.

5. The nucleic acid construct of embodiments 3 or 4, wherein themodified alcohol dehydrogenase exhibits at least a 10 fold increase inactivity with cofactor NADH as compared to an unmodified alcoholdehydrogenase.

6. The nucleic acid construct of embodiment 1, wherein thepolynucleotide shares at least 90% sequence identity with SEQ ID NO:1.

7. The nucleic acid construct of embodiment 1, wherein thepolynucleotide shares at least 95% sequence identity with SEQ ID NO:1.

8. The nucleic acid construct of embodiment 1, wherein thepolynucleotide shares at least 99% sequence identity with SEQ ID NO:1.

9. The nucleic acid construct of embodiment 1, wherein the modifiedalcohol dehydrogenase is a secondary alcohol dehydrogenase.

10. The nucleic acid construct of embodiment 1, wherein the modifiedalcohol dehydrogenase is NADH-dependent.

11. The nucleic acid construct of embodiment 1, wherein the modifiedalcohol dehydrogenase is a microbial alcohol dehydrogenase.

12. The nucleic acid construct of embodiment 11, wherein the microbialalcohol dehydrogenase is a bacterial alcohol dehydrogenase.

13. The nucleic acid construct of embodiment 12, wherein the bacterialalcohol dehydrogenase is a Clostridium beijerinckii alcoholdehydrogenase.

14. A polypeptide sequence comprising a modified alcohol dehydrogenasethat exhibits activity with cofactor NADH as compared to an unmodifiedalcohol dehydrogenase; and wherein the modified alcohol dehydrogenaseshares at least 85% sequence identity with SEQ ID NO:2.

15. The polypeptide sequence of embodiment 14, wherein the modifiedalcohol dehydrogenase exhibits a cofactor preference for NADH overNADPH, as compared to an unmodified alcohol dehydrogenase.

16. The polypeptide sequence of embodiment 14, wherein the modifiedalcohol dehydrogenase exhibits an increased activity for reduction ofacetone to isopropanol with NADH, as compared to an unmodified alcoholdehydrogenase.

17. The polypeptide sequence of embodiment 14, wherein the modifiedalcohol dehydrogenase exhibits an increased activity for oxidation ofisopropanol to acetone, as compared to an unmodified alcoholdehydrogenase.

18. The polypeptide sequence of embodiments 16 or 17, wherein themodified alcohol dehydrogenase exhibits at least a 10 fold increase inactivity with cofactor NADH as compared to an unmodified alcoholdehydrogenase.

19. The polypeptide sequence of embodiment 14, wherein the modifiedalcohol dehydrogenase shares at least 90% sequence identity with SEQ IDNO:2.

20. The polypeptide sequence of embodiment 14, wherein the modifiedalcohol dehydrogenase shares at least 95% sequence identity with SEQ IDNO:2.

21. The polypeptide sequence of embodiment 14, wherein the modifiedalcohol dehydrogenase shares at least 99% sequence identity with SEQ IDNO:2.

22. The polypeptide sequence of embodiment 14, wherein the alcoholdehydrogenase is a secondary alcohol dehydrogenase.

23. The polypeptide sequence of embodiment 14, wherein the modifiedalcohol dehydrogenase is NADH-dependent.

24. The polypeptide sequence of embodiment 14, wherein the modifiedalcohol dehydrogenase is a microbial alcohol dehydrogenase.

25. The polypeptide sequence of embodiment 24, wherein the microbialalcohol dehydrogenase is a bacterial alcohol dehydrogenase.

26. The polypeptide sequence of embodiment 25, wherein the bacterialalcohol dehydrogenase is a Clostridium beijerinckii alcoholdehydrogenase.

27. The polypeptide sequence of embodiment 14, wherein the polypeptidesequence comprises a signal peptide.

28. The polypeptide sequence of embodiment 27, wherein the signalpeptide is a secretion signal.

29. The polypeptide sequence of embodiment 14, wherein the modifiedalcohol dehydrogenase sequence is selected from SEQ ID NOs: 3-38.

30. The polypeptide sequence of embodiment 14, wherein the modifiedalcohol dehydrogenase sequence comprises an amino acid substitution atone or more of the following residues: T38, G198, S199, Y218, I175,I173, R200, P201, C203, G244, E247, T248, K219, G243, Q251, Y267, L294,C295, K340, and K342.

31. The polypeptide sequence of embodiment 14, wherein the modifiedalcohol dehydrogenase sequence comprises an amino acid substitution atresidue T38 with an H amino acid residue.

32. The polypeptide sequence of embodiment 14, wherein the modifiedalcohol dehydrogenase sequence comprises an amino acid substitution atresidue G198 with a D, E, G, K, N, R, S, A, or V amino acid residue.

33. The polypeptide sequence of embodiment 14, wherein the modifiedalcohol dehydrogenase sequence comprises an amino acid substitution atresidue S199 with a D, G, H, I, L, N, R, S, V, C, M, or P amino acidresidue.

34. The polypeptide sequence of embodiment 14, wherein the modifiedalcohol dehydrogenase sequence comprises an amino acid substitution atresidue Y218 with a P, A, D, F, I, N, S, T, V, H, L, or Y amino acidresidue.

35. The polypeptide sequence of embodiment 14, wherein the modifiedalcohol dehydrogenase sequence comprises an amino acid substitution atresidue I173 with a V, T, A, L, F, or Y amino acid residue.

36. The polypeptide sequence of embodiment 14, wherein the modifiedalcohol dehydrogenase sequence comprises an amino acid substitution atresidue I175 with a V or A amino acid residue.

37. The polypeptide sequence of embodiment 14, wherein the modifiedalcohol dehydrogenase sequence comprises an amino acid substitution atresidue R200 with a R, G, S, or N amino acid residue.

38. The polypeptide sequence of embodiment 14, wherein the modifiedalcohol dehydrogenase sequence comprises an amino acid substitution atresidue P201 with a P, E, D, K, or S amino acid residue.

39. The polypeptide sequence of embodiment 14, wherein the modifiedalcohol dehydrogenase sequence comprises an amino acid substitution atresidue C203 with an R or K amino acid residue.

40. The polypeptide sequence of embodiment 14, wherein the modifiedalcohol dehydrogenase sequence comprises an amino acid substitution atresidue G244 with an A, C, M, F, I, W, L, H, or P amino acid residue.

41. The polypeptide sequence of embodiment 14, wherein the modifiedalcohol dehydrogenase sequence comprises an amino acid substitution atresidue E247 with a D, N, Q, or H amino acid residue.

42. The polypeptide sequence of embodiment 14, wherein the modifiedalcohol dehydrogenase sequence comprises an amino acid substitution atresidue T248 with an S, N, A, L, M, or V amino acid residue.

43. The polypeptide sequence of embodiment 14, wherein the modifiedalcohol dehydrogenase sequence comprises one or more of the followingamino acid substitutions: G198E, E247Q, E247D, E247N, Y218F, G198D,I173C, G198L, S199D, Y218P, or Y218A.

44. The polypeptide sequence of embodiment 14, wherein the modifiedalcohol dehydrogenase sequence comprises G198E and E247Q amino acidsubstitutions.

45. The polypeptide sequence of embodiment 14, wherein the modifiedalcohol dehydrogenase sequence comprises a G198E amino acidsubstitution.

46. The polypeptide sequence of embodiment 14, wherein the modifiedalcohol dehydrogenase sequence comprises G198E and Y218F amino acidsubstitutions.

47. The polypeptide sequence of embodiment 14, wherein the modifiedalcohol dehydrogenase sequence comprises G198E and E247D amino acidsubstitutions.

48. The polypeptide sequence of embodiment 14, wherein the modifiedalcohol dehydrogenase sequence comprises G198E and E247N amino acidsubstitutions.

49. The polypeptide sequence of embodiment 14, wherein the modifiedalcohol dehydrogenase sequence comprises G198D and Y218F amino acidsubstitutions.

50. The polypeptide sequence of embodiment 14, wherein the modifiedalcohol dehydrogenase sequence comprises I173C and G198E amino acidsubstitutions.

51. The polypeptide sequence of embodiment 14, wherein the modifiedalcohol dehydrogenase sequence comprises a G198D amino acidsubstitution.

52. The polypeptide sequence of embodiment 14, wherein the modifiedalcohol dehydrogenase sequence comprises G198L and S199D amino acidsubstitutions.

53. The polypeptide sequence of embodiment 14, wherein the modifiedalcohol dehydrogenase sequence comprises G198D and Y218P amino acidsubstitutions.

54. The polypeptide sequence of embodiment 14, wherein the modifiedalcohol dehydrogenase sequence comprises G198E and Y218P amino acidsubstitutions.

55. The polypeptide sequence of embodiment 14, wherein the modifiedalcohol dehydrogenase sequence comprises G198D and Y218A amino acidsubstitutions.

56. The polypeptide sequence of embodiment 14, wherein the modifiedalcohol dehydrogenase is NADH-dependent.

57. A recombinant microorganism comprising the nucleic acid construct ofany one of embodiment 1-13.

58. A method of producing a recombinant microorganism that produces amodified alcohol dehydrogenase that exhibits activity with cofactor NADHas compared to an unmodified alcohol dehydrogenase, the methodcomprising introducing a polynucleotide sequence encoding thepolypeptide sequence of any one of embodiments 14-56 into amicroorganism.

59. The method of embodiment 58, wherein the microorganism is abacterium.

60 The method of embodiment 59, wherein the bacterium is a species ofEscherichia or Bacillus.

61. The method of embodiment 60, wherein the bacterium is Escherichiacoli.

62. The method of embodiment 58, wherein the microorganism is a fungus.

63. The method of embodiment 62, wherein the fungus is a yeast.

64. The method of embodiment 62, wherein the fungus is a species ofSaccharomyces, Pichia or Aspergillus.

65. The method of embodiment 58, wherein the modified alcoholdehydrogenase exhibits a cofactor preference for NADH over NADPH, ascompared to an unmodified alcohol dehydrogenase.

66. A recombinant microorganism expressing a polypeptide sequencecomprising a modified alcohol dehydrogenase that exhibits activity withcofactor NADH as compared to an unmodified alcohol dehydrogenase; andwherein the modified alcohol dehydrogenase shares at least 85% sequenceidentity with SEQ ID NO:2.

67. The recombinant microorganism of embodiment 66, wherein the modifiedalcohol dehydrogenase exhibits a cofactor preference for NADH overNADPH, as compared to an unmodified alcohol dehydrogenase.

68. The recombinant microorganism of embodiment 66, wherein the modifiedalcohol dehydrogenase exhibits an increased activity for reduction ofacetone to isopropanol, as compared to an unmodified alcoholdehydrogenase.

69. The recombinant microorganism of embodiment 66, wherein the modifiedalcohol dehydrogenase exhibits an increased activity for oxidation ofisopropanol to acetone, as compared to an unmodified alcoholdehydrogenase.

70. The recombinant microorganism of embodiments 68 or 69, wherein themodified alcohol dehydrogenase exhibits at least a 10 fold increase inactivity with cofactor NADH as compared to an unmodified alcoholdehydrogenase.

71. The recombinant microorganism of embodiment 66, wherein the modifiedalcohol dehydrogenase shares at least 90% sequence identity with SEQ IDNO:2.

72. The recombinant microorganism of embodiment 66, wherein the modifiedalcohol dehydrogenase shares at least 95% sequence identity with SEQ IDNO:2.

73. The recombinant microorganism of embodiment 66, wherein the modifiedalcohol dehydrogenase shares at least 99% sequence identity with SEQ IDNO:2.

74. The recombinant microorganism of embodiment 66, wherein the alcoholdehydrogenase is a secondary alcohol dehydrogenase.

75. The recombinant microorganism of embodiment 66, wherein the modifiedalcohol dehydrogenase is a microbial alcohol dehydrogenase.

76. The recombinant microorganism of embodiment 75, wherein themicrobial alcohol dehydrogenase is a bacterial alcohol dehydrogenase.

77. The recombinant microorganism of embodiment 76, wherein thebacterial alcohol dehydrogenase is a Clostridium beijerinckii alcoholdehydrogenase.

78. The recombinant microorganism of embodiment 66, wherein thepolypeptide sequence comprises a signal peptide.

79. The recombinant microorganism of embodiment 66, wherein the signalpeptide is a secretion signal.

80. The recombinant microorganism of embodiment 66, wherein the modifiedalcohol dehydrogenase sequence is selected from SEQ ID NOs: 3-38.

81. The recombinant microorganism of embodiment 66, wherein the modifiedalcohol dehydrogenase sequence comprises an amino acid substitution atone or more of the following residues: T38, G198, S199, Y218, I175,I173, R200, P201, C203, G244, E247, T248, K219, G243, Q251, Y267, L294,C295, K340, and K342.

82. The recombinant microorganism of embodiment 66, wherein the modifiedalcohol dehydrogenase sequence comprises an amino acid substitution atresidue T38 with an H amino acid residue.

83. The recombinant microorganism of embodiment 66, wherein the modifiedalcohol dehydrogenase sequence comprises an amino acid substitution atresidue G198 with a D, E, G, K, N, R, S, A, or V amino acid residue.

84. The recombinant microorganism of embodiment 66, wherein the modifiedalcohol dehydrogenase sequence comprises an amino acid substitution atresidue S199 with a D, G, H, I, L, N, R, S, V, C, M, or P amino acidresidue.

85. The recombinant microorganism of embodiment 66, wherein the modifiedalcohol dehydrogenase sequence comprises an amino acid substitution atresidue Y218 with a P, A, D, F, I, N, S, T, V, Y, H, L, or Y amino acidresidue.

86. The recombinant microorganism of embodiment 66, wherein the modifiedalcohol dehydrogenase sequence comprises an amino acid substitution atresidue I173 with a V, T, A, L, T, F, or Y amino acid residue.

87. The recombinant microorganism of embodiment 66, wherein the modifiedalcohol dehydrogenase sequence comprises an amino acid substitution atresidue I175 with a V or A amino acid residue.

88. The recombinant microorganism of embodiment 66, wherein the modifiedalcohol dehydrogenase sequence comprises an amino acid substitution atresidue R200 with a R, G, S, or N amino acid residue.

89. The recombinant microorganism of embodiment 66, wherein the modifiedalcohol dehydrogenase sequence comprises an amino acid substitution atresidue P201 with a P, E, D, K, or S amino acid residue.

90. The recombinant microorganism of embodiment 66, wherein the modifiedalcohol dehydrogenase sequence comprises an amino acid substitution atresidue C203 with an R or K amino acid residue.

91. The recombinant microorganism of embodiment 66, wherein the modifiedalcohol dehydrogenase sequence comprises an amino acid substitution atresidue G244 with an A, C, M, F, I, W, L, H, or P amino acid residue.

92. The recombinant microorganism of embodiment 66, wherein the modifiedalcohol dehydrogenase sequence comprises an amino acid substitution atresidue E247 with a D, N, Q, or H amino acid residue.

93. The recombinant microorganism of embodiment 66, wherein the modifiedalcohol dehydrogenase sequence comprises an amino acid substitution atresidue T248 with an S, N, A, L, M, or V amino acid residue.

94. The recombinant microorganism of embodiment 66, wherein the modifiedalcohol dehydrogenase sequence comprises one or more of the followingamino acid substitutions: G198E, E247Q, E247D, E247N, Y218F, G198D,I173C, G198L, S199D, Y218P, or Y218A.

95. The recombinant microorganism of embodiment 66, wherein the modifiedalcohol dehydrogenase sequence comprises G198E and E247Q amino acidsubstitutions.

96. The recombinant microorganism of embodiment 66, wherein the modifiedalcohol dehydrogenase sequence comprises a G198E amino acidsubstitution.

97. The recombinant microorganism of embodiment 66, wherein the modifiedalcohol dehydrogenase sequence comprises G198E and Y218F amino acidsubstitutions.

98. The recombinant microorganism of embodiment 66, wherein the modifiedalcohol dehydrogenase sequence comprises G198E and E247D amino acidsubstitutions.

99. The recombinant microorganism of embodiment 66, wherein the modifiedalcohol dehydrogenase sequence comprises G198E and E247N amino acidsubstitutions.

100. The recombinant microorganism of embodiment 66, wherein themodified alcohol dehydrogenase sequence comprises G198D and Y218F aminoacid substitutions.

101. The recombinant microorganism of embodiment 66, wherein themodified alcohol dehydrogenase sequence comprises I173C and G198E aminoacid substitutions.

102. The recombinant microorganism of embodiment 66, wherein themodified alcohol dehydrogenase sequence comprises a G198D amino acidsubstitution.

103. The recombinant microorganism of embodiment 66, wherein themodified alcohol dehydrogenase sequence comprises G198L and S199D aminoacid substitutions.

104. The recombinant microorganism of embodiment 66, wherein themodified alcohol dehydrogenase sequence comprises G198D and Y218P aminoacid substitutions.

105. The recombinant microorganism of embodiment 66, wherein themodified alcohol dehydrogenase sequence comprises G198E and Y218P aminoacid substitutions.

106. The recombinant microorganism of embodiment 66, wherein themodified alcohol dehydrogenase sequence comprises G198D and Y218A aminoacid substitutions.

107. The recombinant microorganism of embodiment 66, wherein themodified alcohol dehydrogenase is NADH-dependent.

While the disclosure has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the disclosure following, in general, theprinciples of the disclosure and including such departures from thepresent disclosure as come within known or customary practice within theart to which the disclosure pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

While the disclosure is divided into sections, subsections, and furtherdelineations, this is simply for exemplary purposes and is in no wayintended to limit the methods, processes, compositions, products,substrates, media, and the like for use in any other aspect of thedisclosure. For example, the disclosure of substrates used in thefermentation phase subsection does not limit the use of the substratesto fermentation phase processes.

All references, articles, publications, patents, patent publications,and patent applications cited herein are incorporated by reference intheir entireties for all purposes.

However, mention of any reference, article, publication, patent, patentpublication, and patent application cited herein is not, and should notbe taken as, an acknowledgment or any form of suggestion that theyconstitute valid prior art or form part of the common general knowledgein any country in the world.

1. A nucleic acid construct comprising a polynucleotide sequenceencoding a modified alcohol dehydrogenase, wherein the alcoholdehydrogenase exhibits activity with cofactor NADH as compared to anunmodified alcohol dehydrogenase; and wherein the polynucleotidesequence shares at least 85% sequence identity with SEQ ID NO:
 1. 2. Thenucleic acid construct of claim 1, wherein the modified alcoholdehydrogenase exhibits a cofactor preference for NADH over NADPH, ascompared to an unmodified alcohol dehydrogenase.
 3. The nucleic acidconstruct of claim 1, wherein the modified alcohol dehydrogenaseexhibits an increased activity for reduction of acetone to isopropanolwith NADH, as compared to an unmodified alcohol dehydrogenase.
 4. Thenucleic acid construct of claim 1, wherein the modified alcoholdehydrogenase exhibits an increased activity for oxidation ofisopropanol to acetone, as compared to an unmodified alcoholdehydrogenase.
 5. A method of producing a recombinant microorganism thatproduces a modified alcohol dehydrogenase that exhibits activity withcofactor NADH as compared to an unmodified alcohol dehydrogenase, themethod comprising introducing a polynucleotide sequence encoding apolypeptide sequence into a microorganism; wherein the polypeptidesequence comprises a modified alcohol dehydrogenase that exhibitsactivity with cofactor NADH as compared to an unmodified alcoholdehydrogenase; and wherein the modified alcohol dehydrogenase shares atleast 85% sequence identity with SEQ ID NO:
 2. 6. The method of claim 5,wherein the microorganism is a bacterium or a fungus.
 7. The method ofclaim 5, wherein the modified alcohol dehydrogenase exhibits a cofactorpreference for NADH over NADPH, as compared to an unmodified alcoholdehydrogenase.
 8. A recombinant microorganism expressing a polypeptidesequence comprising a modified alcohol dehydrogenase that exhibitsactivity with cofactor NADH as compared to an unmodified alcoholdehydrogenase; and wherein the modified alcohol dehydrogenase shares atleast 85% sequence identity with SEQ ID NO:
 2. 9. The recombinantmicroorganism of claim 8, wherein the modified alcohol dehydrogenaseexhibits a cofactor preference for NADH over NADPH, as compared to anunmodified alcohol dehydrogenase.